The present invention relates to antigen-specific down-regulation of an immune response using Zot or zonulin. Specifically, the present invention provides a method for inhibiting antigen presenting cell-mediated antigen-specific lymphocyte proliferation in a dose-dependent manner by administering an effective amount of Zot or zonulin.
The tight junctions (hereinafter xe2x80x9ctjxe2x80x9d) or zonula occludens (hereinafter xe2x80x9cZOxe2x80x9d) are one of the hallmarks of absorptive and secretory epithelia (Madara, J. Clin. Invest., 83:1089-1094 (1989); and Madara, Textbook of Secretory Diarrhea Eds. Lebenthal et al, Chapter 11, pages 125-138 (1990). As a barrier between apical and basolateral compartments, they selectively regulate the passive diffusion of ions and water-soluble solutes through the paracellular pathway (Gumbiner, Am. J. Physiol., 253(Cell Physiol. 22):C749-C758 (1987)). This barrier maintains any gradient generated by the activity of pathways associated with the transcellular route (Diamond, Physiologist, 20:10-18 (1977)).
There is abundant evidence that ZO, once regarded as static structures, are in fact dynamic and readily adapt to a variety of developmental (Magnuson et al, Dev. Biol., 67:214-224 (1978); Revel et al, Cold Spring Harbor Symp. Quant. Biol., 40:443-455 (1976); and Schneeberger et al, J. Cell Sci., 32:307-324 (1978)), physiological (Gilula et al, Dev. Biol., 50:142-168 (1976); Madara et al, J. Membr. Biol., 100:149-164 (1987); Mazariegos et al, J. Cell Biol., 98:1865-1877 (1984); and Sardet et al, J. Cell Biol., 80:96-117 (1979)), and pathological (Milks et al, J. Cell Biol., 103:2729-2738 (1986); Nash et al, Lab. Invest., 59:531-537 (1988); and Shasby et al, Am. J. Physiol., 255(Cell Physiol., 24):C781-C788 (1988)) circumstances. The regulatory mechanisms that underlie this adaptation are still not completely understood. However, it is clear that, in the presence of Ca2+, assembly of the ZO is the result of cellular interactions that trigger a complex cascade of biochemical events that ultimately lead to the formation and modulation of an organized network of ZO elements, the composition of which has been only partially characterized (Diamond, Physiologist, 20:10-18 (1977)). A candidate for the transmembrane protein strands, occluding, has been identified (Furuse et al, J. Membr. Biol., 87:141-150 (1985)).
Six proteins have been identified in a cytoplasmic submembranous plaque underlying membrane contacts, but their function remains to be established (Diamond, supra). ZO-1 and ZO-2 exist as a heterodimer (Gumbiner et al, Proc. Natl. Acad. Sci., USA, 88:3460-3464 (1991)) in a detergent-stable complex with an uncharacterized 130 kD protein (ZO-3). Most immunoelectron microscopic studies have localized ZO-1 to precisely beneath membrane contacts (Stevenson et al, Molec. Cell Biochem., 83:129-145 (1988)). Two other proteins, cingulin (Citi et al, Nature (London), 333:272-275 (1988)) and the 7H6 antigen (zhong et al, J. Cell Biol., 120:477-483 (1993)) are localized further from the membrane and have not yet been cloned. Rab 13, a small GTP binding protein has also recently been localized to the junction region (Zahraoui et al, J. Cell Biol., 124:101-115 (1994)). Other small GTP-binding proteins are known to regulate the cortical cytoskeleton, i.e., rho regulates actin-membrane attachment in focal contacts (Ridley et al, Cell, 70:389-399 (1992)), and rac regulates growth factor-induced membrane ruffling (Ridley et al, Cell, 70:401-410 (1992)). Based on the analogy with the known functions of plaque proteins in the better characterized cell junctions, focal contacts (Guan et al, Nature, 3:690-692 (1992)), and adherens junctions (Tsukita et al, J. Cell Biol., 1:1049-1053 (1993)), it has been hypothesized that tj-associated plaque proteins are involved in transducing signals in both directions across the cell membrane, and in regulating links to the cortical actin cytoskeleton.
To meet the many diverse physiological and pathological challenges to which epithelia are subjected, the ZO must be capable of rapid and coordinated responses that require the presence of a complex regulatory system. The precise characterization of the mechanisms involved in the assembly and regulation of the ZO is an area of current active investigation.
There is now a body of evidence that tj structural and functional linkages exist between the actin cytoskeleton and the tj complex of absorptive cells (Gumbiner et al, supra; Madara et al, supra; and Drenchahn et al, J. Cell Biol., 107:1037-1048 (1988)). The actin cytoskeleton is composed of a complicated meshwork of microfilaments whose precise geometry is regulated by a large cadre of actin-binding proteins. An example of how the state of phosphorylation of an actin-binding protein might regulate cytoskeletal linking to the cell plasma membrane is the myristoylated alanine-rich C kinase substrate (hereinafter xe2x80x9cMARCKSxe2x80x9d). MARCKS is a specific protein kinase C (hereinafter xe2x80x9cPKCxe2x80x9d) substrate that is associated with the cytoplasmic face of the plasma membrane (Aderem, Elsevier Sci. Pub. (UK), pages 438-443 (1992)). In its non-phosphorylated form, MARCKS crosslinks to the membrane actin. Thus, it is likely that the actin meshwork associated with the membrane via MARCKS is relatively rigid (Hartwig et al, Nature, 356:618-622 (1992)). Activated PKC phosphorylates MARCKS, which is released from the membrane (Rosen et al, J. Exp. Med., 172:1211-1215 (1990); and Thelen et al, Nature, 351:320-322 (1991)). The actin linked to MARCKS is likely to be spatially separated from the membrane and be more plastic. When MARCKS is dephosphorylated, it returns to the membrane where it once again crosslinks actin (Hartwig et al, supra; and Thelen et al, supra). These data suggest that the F-actin network may be rearranged by a PKC-dependent phosphorylation process that involves actin-binding proteins (MARCKS being one of them).
Most Vibrio cholerae vaccine candidates constructed by deleting the ctxa gene encoding cholera toxin (CT) are able to elicit high antibody responses, but more than one-half of the vaccinee still develop mild diarrhea (Levine et al, Infect. Immun., 56(1):161-167 (1988)). Given the magnitude of the diarrhea induced in the absence of CT, it was hypothesized that V. cholerae produce other enterotoxigenic factors, which are still present in strains deleted of the ctxa sequence (Levine et al, supra). As a result, a second toxin, zonula occludens toxin (hereinafter xe2x80x9cZot) elaborated by V. cholerae, and which contribute to the residual diarrhea, was discovered (Fasano et al, Proc. Nat. Acad. Sci., USA, 8:5242-5246 (1991)). The zot gene is located immediately adjacent to the ctx genes. The high percent concurrence of the zot gene with the ctx genes among V. cholerae strains (Johnson et al, J. Clin. Microb., 31/3:732-733 (1993); and Karasawa et al, FEBS Microbiology Letters, 106:143-146 (1993)) suggests a possible synergistic role of Zot in the causation of acute dehydrating diarrhea typical of cholera. Recently, the zot gene has also been identified in other enteric pathogens (Tschape, 2nd Asian-Pacific Symposium on Typhoid fever and other Salmonellosis, 47(Abstr.) (1994)).
It has been previously found that, when tested on rabbit ileal mucosa, Zot increases the intestinal permeability by modulating the structure of intercellular tight junctions (Fasano et al, supra). It has been found that as a consequence of modification of the pericellular pathway, the intestinal mucosa becomes more permeable. It also was found that Zot does not affect Na+-glucose coupled active transport, is not cytotoxic, and fails to completely abolish the transepithelial resistance (Fasano et al, supra).
More recently, it has been found that Zot is capable of reversibly opening tight junctions in the intestinal mucosa, and thus Zot, when co-administered with a therapeutic agent, is able to effect intestinal delivery of the therapeutic agent, when employed in an oral dosage composition for intestinal drug delivery (WO 96/37196, U.S. Pat. No. 5,827,534 and U.S. Pat. No. 5,665,389; each of which is incorporated by reference herein in their entirety). It has also been found that Zot is capable of reversibly opening tight junctions in the nasal mucosa, and thus Zot, when co-administered with a therapeutic agent, is able to enhance nasal absorption of a therapeutic agent (WO 98/30211 and U.S. Pat. No. 5,908,825; which is incorporated by reference herein in its entirety).
In U.S. Pat. No. 5,864,014 and U.S. Pat. No. 5,912,323; which are incorporated by reference herein in their entirety, Zot receptors from CaCo2 cells, heart, intestinal and brain tissue has been identified and isolated. The Zot receptors represent the first step of the pericellular pathway involved in the regulation of epithelial intestinal and nasal permeability.
In U.S. Pat. No. 5,945,510, which is incorporated by reference herein in its entirety, mammalian proteins that are immunologically and functionally related to Zot, and that function as the physiological modulator of mammalian tight junctions, have been identified and purified. These mammalian proteins, referred to as xe2x80x9czonulinxe2x80x9d, are useful for enhancing absorption of therapeutic agents across tight junctions of intestinal and nasal mucosa, as well as across tight junctions of the blood brain barrier. These proteins are further characterized by the ability to bind to the Zot receptors.
In pending U.S. patent application Ser. No. 09/127,815 filed Aug. 3, 1998, entitled xe2x80x9cPeptide Antagonists of Zonulin and Methods for Use of the Samexe2x80x9d, which is incorporated by reference herein in its entirety, peptide antagonists of zonulin have been identified. Said peptide antagonists bind to Zot receptor, yet do not function to physiologically modulate the opening of mammalian tight junctions. The peptide antagonists competitively inhibit the binding of Zot and zonulin to the Zot receptor, thereby inhibiting the ability of Zot and zonulin to physiologically modulate the opening of mammalian tight junctions.
For a complete discussion of immune responses and immunomodulation, see Chapter 10 xe2x80x9cRecent Advances in Immunologyxe2x80x9d, by Sztein et al, New Generation of Vaccines, pages 99-125, Eds. Levine et al (1997), the disclosure of which is hereby incorporated by reference.
One of the primary mechanisms of protection against infectious agents involves specific or acquired immunity. In contrast to innate immunity, the effector mechanisms of acquired immunity that include, among others, antibodies, cytotoxic lymphocytes (hereinafter xe2x80x9cCTLxe2x80x9d), T lymphocyte-derived cytokines (such as IFN-xcex3, IL-4, etc.) are induced following exposure to antigens or infectious agents and increase in magnitude with successive exposures to the specific antigens. This ability to xe2x80x9crecallxe2x80x9d previous exposures to antigens and respond rapidly with immunological effector responses of increased magnitude (immunologic memory) constitutes the foundation for immunoprophylactic vaccination against infectious agents. The chief cell types involved in specific immune responses are T and B lymphocytes.
B lymphocytes or B cells are derived from the bone marrow and are the precursors of antibody secreting cells (plasma cells). B cells recognize antigens (proteins, carbohydrates or simple chemical groups) through immunoglobulin receptors on the cell membrane (Fearon et al, Science, 272:50-53 (1996); ziegler-Heitbroack et al, Immunol. Today, 14:121-152 (1993); and Banchereau et al, Adv. Immunol., 52;125-262 (1992)). After triggering by antigen, they clonally expand and switch their expression of antibody isotype (e.g., IgM to IgG, IgE or IgA) under the influence of cytokines derived from T cells, macrophages and other cell types. Somatically-mutated, high affinity B cells are generated and selected by antigen in and around the germinal centers that are formed in lymph nodes, spleen, Peyers"" patches and more disorganized lymphatic aggregates of the peripheral lymphoid system (Banchereau et al, (1996) supra; Clark et al, Ann. Rev. Immunol., 9:97-127 (1991); and MacLennan et al, Immunol. Today, 14:29-34 (1993)). They are the basis for B cell memory.
T lymphocytes or T cells, in contrast to B cells, recognize peptides derived from protein antigens that are presented on the surface of antigen presenting cells (hereinafter xe2x80x9cAPCxe2x80x9d) in conjunction with Class I or Class II major histocompatibility complex (MHC) molecules. Clones of T lymphocytes expressing T cell receptors (hereinafter xe2x80x9cTCRxe2x80x9d) of appropriate affinity are triggered by antigen to proliferate and develop into effector cells (Fearon, (1996) supra; Sprent et al Cell, 76:315-322 (1994); and Hendrick et al, Germain, Fundamental Immunology, 3rd ed., pages 629-676 (1993)). After elimination of the infectious agent, the antigen-specific clones remain as memory T cells that, upon subsequent exposures to antigen, provide a stronger, more rapid and sometimes qualitatively different specific immune response.
There are two main populations of T cells, those expressing CD4 molecules and those expressing CD8 molecules. CD4 and CD8 molecules are T cell surface. glycoproteins that serve as important accessory molecules (co-receptors) during antigen presentation by binding to Class II and Class I MHC molecules, respectively (Hendrick et al, supra (1993)). Thus, CD4 and CD8 molecules play a significant role in stabilizing the interactions of T cells and APC initiated by the specific binding of the TCR complex to antigenic peptides presented in association with MHC molecules. Consequently, CD4 and CD8 molecules, originally used primarily as markers to identify T cell populations with different functional characteristics, play a major role in Class II MHC-restricted and Class I MHC-restricted T cell activation. CD4+ cells (T helper or Th) are mainly involved in inflammatory responses and providing help for antibody production by B cells, while CD8+ cells (T cytotoxic or Tc) compose the majority of xc2x0 CTL primarily involved in Class I MHC-restricted killing of target cells infected by pathogenic organisms, including bacteria, viruses and parasites (Sztein et al, J. Immunol., 155:3987-3993 (1995); Kaufman, Ann. Rev. Immunol., 11:129-163 (1993) and Immunol. Today, 9:168-174 (1988); Townsend et al, Cell, 44:959-968 (1986); Malik et al, Proc. Natl. Acad. Sci., USA, 88:3300-3304 (1991); Sedegah et al, J. Immunol., 1:966-971 (1992); and Shearer et al, Immunol. Today, 17:21-24 (1996)).
Successful antigen specific activation of T cells resulting in T cell expansion and differentiation (or lymphocyte proliferation) requires a first signal provided by the interaction of TCR on the surface of T cells with MHC-antigen complexes on APC and a second, complementary, signal provided by soluble factors, such as IL-2, or binding of CD28 (a co-stimulatory molecule) to members of the B7 family (e.g., CD80 (B7-1) or CD86 (B7-2)) on APC (Lenschow, Ann. Rev. Immunol., 14:233-258(1996); and Linsley et al, Ann. Rev. Immunol., 11:191-212(1993)). The study of the CD28/B7 co-stimulatory pathway and other adhesion molecules that help stabilize T cell-APC interactions (and which also appear to play critical in roles in lymphocyte homing), is one of the key areas in which many significant advances have been made in recent years.
Presentation of antigens to T cells involves a series of intracellular events within the APC, including the generation of antigenic peptide fragments, binding of these peptides to MHC molecules to form stable peptide-MHC complexes and transport of these complexes to the cell surface where they can be recognized by TCR in the surface of T cells. Evidence has accumulated for the existence of two main pathways of antigen processing and presentation (xe2x80x9cclassical pathwaysxe2x80x9d). One of these pathways, the xe2x80x9ccytosolic pathwayxe2x80x9d, is predominantly used for presentation of peptides produced endogenously in the APC, such as viral proteins, tumor antigens and self-peptides, associated with Class I MHC molecules (Hendrick et al, supra; and Germain, supra (1993)). The presentation of large numbers of self-peptides complexed to Class I MHC molecules results from the inability of APC to differentiate between self and non-self. Under normal conditions, most T cells selected to recognize self-peptides are eliminated during T cell differentiation or are actively down regulated, and consequently can not be activated by self-peptide-Class I MHC complexes. The second xe2x80x9cclassical pathwayxe2x80x9d of antigen processing and presentation, xe2x80x9cendosomal pathwayxe2x80x9d, which is predominantly used for presentation of soluble exogenous antigens bound to Class II MHC molecules, involves the capture of antigen by APC, either by binding to a specific receptor or by uptake in the fluid phase by macropinocytosis (Lanzavecchia, Curr. Opin. Immunol., 8:348-354 (1996)). Triggering of T cells through the TCR has been shown with as few as 200-600 peptide/MHC complexes in the case of influenza nucleoproteins (Falk et al, Semin. Immunol., 5:81-94 (1993)). In most immune responses, antigenic epitopes associated with Class I MHC molecules trigger the activation of CD8+ CTL responses, while antigenic fragments (epitopes) derived from soluble proteins complexed to Class II MHC molecules are recognized by CD4+ Th cells. These findings are among the most important contributions made over the past few years on the mechanisms involved in the early stages of immune activation and are critical for the development of successful vaccines.
As mentioned above, there are two xe2x80x9cclassicalxe2x80x9d pathways of antigen processing and presentation. The Class I MHC pathway is that most commonly used for processing of cellular proteins present in most, if not all, cellular compartments, including the cytosol, nucleus and mitochondria (Falk et al, supra (1993)) for recognition by CD8+ CTL. The Class II MHC pathway is predominantly used for processing and presentation of exogenous antigens, such as proteins produced by extracellular bacteria and other infectious microorganisms that can be presented to CD4+ Th cells. Both Class I and II MHC molecules bind peptide antigens through the use of surface If xe2x80x9creceptorsxe2x80x9d or xe2x80x9cbinding cleftsxe2x80x9d. However, the route of antigen processing and preparation varies dramatically between the two. Class I antigens are processed and prepared by the xe2x80x9ccytosolic pathwayxe2x80x9d. Specifically, peptides synthesized intracellularly are degraded into small protein fragments which are then carried across the membrane of the endoplasmic reticulum (ER). Inside the ER, antigenic fragments bind to Class I MHC molecules forming a complex that is then transported to the Golgi apparatus and ultimately to the cell surface where they are recognized by TCR, signalling antigen-specific CTL expansion and differentiation, the first step of an immune response. Class II antigens, on the other hand, are processed and prepared by the xe2x80x9cendosomal pathwayxe2x80x9d. Specifically, native antigens are captured by a circulating APC, the antigen binding to a specific or nonspecific receptor. The antigen is then internalized by the APC by a mechanism of receptor-mediated endocytosis or pinocytosis. The internalized antigen is then localized in an endosome, a membrane bound vesicle involved in the intracellular transport and degradation of the antigen. Cleaved peptide fragments then bind to Class II MHC molecules to form a complex that is transported through the Golgi apparatus, into the endosomal compartment, and to the cell surface to become recognized by TCR, again signaling the antigen-specific Th cell expansion and differentiation.
APC play a vital role in the generation of an immune response. For presentation of processed antigens to CTL in a Class I-restricted fashion, the APC must express Class I MHC molecules and have the ability to express on the cell surface endogenously produced proteins complexed to Class I MHC molecules. Almost all cells endogenously producing viral, parasitic, or bacterial proteins or tumor antigens that gain access to the cytosol can function as APC. For presentation of processed antigens to Th cells in a Class II restricted fashion, the APC must be able to recognize and bind the antigen through specific or nonspecific receptors for the particular antigen. Cells that most efficiently present antigens to Th lymphocytes, so called professional APC include dendritic cells (DC), macrophages, B lymphocytes, Langerhans cells, and, in certain instances, human endothelial cells (Lanzavecchia, supra (1996)).
DC that originate in the bone marrow are considered to be the most efficient APC for presentation of soluble antigens. DC capture antigens on the periphery and migrate to the spleen or lymph nodes, where they efficiently activate the Th cells, particularly naive T cells (Lanzavecchia, supra (1996); and Peters et al, Immunol. Today, 17:273-278 (1996)). Several unique characteristics enable DC to function so effectively as antigen presenters. Specifically, they have the ability to internalize soluble antigens by several mechanisms, including constitutive macropinocytosis, internalization of antigen-antibody complexes through CD32 receptor binding, and internalization of mannosylated or fucosylated antigens through mannose receptor binding. This allows DC to sample large amounts of fluid in short periods of time, accumulating them in a lysosomal compartment containing Class II MHC molecules and proteases. DC also constitutively express a number of costimulatory and other adhesion molecules that are upregulated by proinflammatory cytokines such as IL-1xcex1, IL-1xcex2, and TNF-xcex1, thereby enhancing their ability to function as APC for Class II MHC restricted Th immune responses.
Macrophages and other mononuclear phagocytes are probably the most effective APC for antigens derived from most pathogenic microorganisms other than viruses through their ability to phagocytose large particles, such as bacteria and parasites. Under typical conditions, phagocytized microorganisms are then killed in the phagolysosomes and digested, resulting in the generation of antigenic fragments available for binding to Class II MHC molecules for presentation to Th cells. Other important mechanisms that allow macrophages to serve as effective APC include their ability to internalize soluble antigens through binding of antigen-antibody complexes to CD16, CD32 and CD64 receptors. Macrophages, also internalize complement coated proteins through receptors for C3 and other Cxe2x80x2 components and upon stimulation by growth factors, by macropinocytosis. Moreover, macrophages express receptors for mannose and are a major source of pro-inflammatory cytokines including IL-1xcex1, IL-1xcex2, IL-6, IL-8, IL-12, TNF-xcex1, and TNF-xcex2 that exert potent immunoregulatory activities on T cell responses (Sztein et al, supra (1997)).
B lymphocytes are very effective APC for soluble antigens for presentation to Th cells. This is largely based on their ability to bind and internalize specific soluble antigens very efficiently through the B-cell receptor complex (BCR), consisting of the specific membrane immunoglobin (mIg) and the Iga (CD79xcex1)-Igxcex2 (CD79xcex2) heterodimer (Falk et al, supra (1993)).
Langerhans cells (LC), derived from bone marrow progenitors, are considered to be the only cells present in the epidermis with APC capabilities. LC migrate out of the epidermis via the lymphatics to the regional lymph nodes where they develop into DC. Interestingly, LC express CD1, a nonclassical MHC molecule capable of presenting to T cells, in a restricted fashion, nonprotein antigens such as microbial lipid and glycolipid antigens.
The invention herein focuses on the antigen specific down-regulation of APC-mediated immune responses. The invention stems from the discovery of a macrophage surface receptor to which Zot binds in a specific and saturable way. The present invention describes a method for using Zot or zonulin as antigen-specific immunoregulators and in immunotherapeutics. Specifically, both Zot and zonulin inhibit APC-mediated antigen-specific lymphocyte proliferation in a dose dependent manner without affecting mitogen induced responses. This down-regulation of the immune response is at least in part associated with the decreased uptake of antigen.
Currently available modulators of immune responses, such as cyclosporin and steroidal compounds, have a generalized effect on antigen and mitogen stimulations of the immune system (Reed et al, J. Immunol., 17:150-154 (1986)). The invention disclosed herein offers the advantage of enabling the down-regulation of immune responses to a particular antigen without inducing negative side effects, such as increased susceptibility to infection and generalized immune suppression, typical of the immunomodulators of the prior art.
It is a object of the invention to provide a method for down-regulating an animal host""s immune response to certain antigens, thereby facilitating immune based therapies. Specifically, it is an object of the invention to inhibit the ability of antigen presenting cells (APC) to process and present antigens to lymphocytes, thereby suppressing the lymphocyte proliferation and subsequent immune system reactions in response to defined antigens.
It is a further object of the invention to provide a treatment for an animal afflicted with an autoimmune or immune related disease or disorder such as multiple sclerosis, rheumatoid arthritis, insulin dependent diabetes mellitus, celiac disease, Sjogren""s syndrome, systemic lupus erythematsosus, auto-immune thyroiditis, idiopathic thrombocytopenic purpura, hemolytic anemia, Grave""s disease, Addison""s disease, autoimmune orchitis, pernicious anemia, vasculitis, autoimmune coagulopathies, myasthenia gravis, polyneuritis, pemphigus, rheumatic carditis, polymyositis, dermatomyositis, and scleroderma by administering an effective amount of a Zot-related immunoregulator. In an alternative embodiment, the treatment of the animal afflicted with an autoimmune or immune related disease or disorder may involve the administration of an effective amount of a Zot-related immunoregulator in combination with a specific auto-immune related antigen(s).
It is a further object of the invention to provide a treatment of an animal afflicted with immune rejection subsequent to tissue or organ transplantation by administering an effective amount of a Zot-related immunoregulator. In an alternative embodiment, the treatment of the animal afflicted with immune rejection subsequent to tissue or organ transplantation may involve the administration of an effective amount of a Zot-related immunoregulator in combination with a specific transplantation antigen(s).
It is a further object of the invention to provide a treatment for an animal afflicted with an inflammatory or allergic disease or disorder such as asthma, psoriasis, eczematous dermatitis, Kaposi""s sarcoma, multiple sclerosis, inflammatory bowel disease, proliferative disorders of smooth muscle cells, and inflammatory conditions associated with mycotic, viral, parasitic, or bacterial infections by administering a therapeutically effective amount of a Zot-related immunoregulator. In an alternative embodiment, the treatment of the animal afflicted with an inflammatory or allergic disease or disorder may involve the administration of an effective amount of a Zot-related immunoregulator in combination with a specific inflammatory related antigen(s) or allergen(s).